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Real-time P2X7-dependent intracellular potassium dynamics observed with KS6. (a) Kinetic trace of potassium efflux from J774A.1 cells stimulated with 5 mM ATP at the indicated time point in the presence of 0 mM additional KCl (normal DMEM medium), 50 or 130 mM additional extracellular KCl. Traces represent the mean and standard error of 10–20 cells in each field. (b) Response at 40 min of potassium efflux (top panel) or TO-PRO-3 uptake (bottom panel) of J774A.1 cells primed for 4 h with 1 μg/ml LPS and treated with 1, 3 or 5 mM extracellular ATP. Bars represent mean and standard deviation of 20 cells in each condition. Statistics were performed by one-way ANOVA with Fischer’s LSD comparison test. *Indicates P<0.05 and **** indicates P<0.0001. (c) Representative fields at the indicated time points of LPS-primed J774A.1 loaded with KS6 (red) and treated with 1, 3 or 5 mM extracellular ATP in the presence of TO-PRO-3 (cyan). Scale bar represents 50 μM. Pre-treatment of LPS-primed, ATP-stimulated J774A.1 macrophages with the P2X7 inhibitor A438079 suppresses (d) potassium efflux (e) and membrane permeability. Traces represent mean and standard error for five representative cells. Results are representative of at least two experiments

Real-time P2X7-dependent intracellular potassium dynamics observed with KS6. (a) Kinetic trace of potassium efflux from J774A.1 cells stimulated with 5 mM ATP at the indicated time point in the presence of 0 mM additional KCl (normal DMEM medium), 50 or 130 mM additional extracellular KCl. Traces represent the mean and standard error of 10–20 cells in each field. (b) Response at 40 min of potassium efflux (top panel) or TO-PRO-3 uptake (bottom panel) of J774A.1 cells primed for 4 h with 1 μg/ml LPS and treated with 1, 3 or 5 mM extracellular ATP. Bars represent mean and standard deviation of 20 cells in each condition. Statistics were performed by one-way ANOVA with Fischer’s LSD comparison test. *Indicates P<0.05 and **** indicates P<0.0001. (c) Representative fields at the indicated time points of LPS-primed J774A.1 loaded with KS6 (red) and treated with 1, 3 or 5 mM extracellular ATP in the presence of TO-PRO-3 (cyan). Scale bar represents 50 μM. Pre-treatment of LPS-primed, ATP-stimulated J774A.1 macrophages with the P2X7 inhibitor A438079 suppresses (d) potassium efflux (e) and membrane permeability. Traces represent mean and standard error for five representative cells. Results are representative of at least two experiments

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P2X7 purinergic receptor engagement with extracellular ATP induces transmembrane potassium and calcium flux resulting in assembly of the NLRP3 inflammasome in LPS-primed macrophages. The role of potassium and calcium in inflammasome regulation is not well understood, largely due to limitations in existing methods for interrogating potassium in real...

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... It was observed that Nano-spheres, ellipses, and rods restrained the intracellular calcium in iBMDM cells similar to the LPS and Nigericin (positive control) treated iBMDMs. Potassium efflux is associated with calcium influx through the cell membrane channel 75 . Nigericin is a known NLRP3 signal 2 molecule that increases potassium efflux through the cellular membrane, resulting in calcium influx upstream of NLRP3 inflammasomes. ...
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... As observed during PLC inhibition, EGTA chelation of extracellular Ca 2+ significantly reduced LTB 4 production compared to untreated BMNs ( Fig 4A; p�0.0001). Influx of extracellular Ca 2+ also requires the cell to maintain a membrane potential by efflux of intracellular potassium (K + ) [67,68]. Therefore, if extracellular Ca 2+ is required, disrupting the K + gradient should also inhibit LTB 4 synthesis. ...
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... This allosteric site is ideal to facilitate druggability of the P2X7 receptor, as allosteric sites are commonly more selective, allow lower target-based toxicity, fewer side effects, and accessible physicochemical properties (43,44). We previously investigated the inhibitory function of A438079 and reported a potent ability to ameliorate extracellular ATP-evoked potassium and calcium flux and subsequent inflammasome activation in mouse macrophages (26). We performed UV-VIS spectrophotometric analysis of A438079 in PBS ( Figure 2B) and identified two diagnostic absorption peaks at 226 nm and 258 nm which exhibit linear response from 40 µM to 2500 µM ( Figure 2C). ...
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... The former, induced by lipopolysaccharide (LPS), facilitates mRNA expression of Nlrp3 and Il1β through NF-κB pathway. The latter, stimulated with extracellular ATP, pore-forming toxins, etc., thereby drives mitochondrial (mt) dysfunction [24,25] and concurrently impairs ion homeostasis [26,27], resulting in the assembly of NLRP3 components, namely NLRP3 inflammasome activation. According to earlier reports, Dioscin suppresses NF-κB activation [20] and mtROS generation [28]. ...
... K + efflux and excessive generation of ROS could trigger the assembly of NLRP3 inflammasome [43][44][45]. Earlier literature documented that K + efflux facilitated mtROS generation in BMDMs [26,46]. Therefore, the influence of K + efflux on NLRP3 activation and the regulatory role of Dioscin in K + efflux was further investigated. ...
... A prior investigation concluded that mtROS generation could be enhanced by impaired ion homeostasis [55]. Interestingly, recent studies reported that K + efflux is implicated in mtROS production [26,46,56]. Specifically, K + efflux could activate NLRP3 inflammasome and augment IL-1β secretion [43,46,57]. ...
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... The above changes will further promote the activation step. In the activation step, some activators, including adenosine triphosphate (ATP), protoxin, viral RNA, and particulate matter [36], cause changes in intracellular ion flow [37][38][39][40][41][42][43], mitochondrial dysfunction [44][45][46], and lysosomal rupture [47][48][49][50]. Then, the oligomerization and activation of NLRP3 inflammasome start. ...
... The priming stage aims to upregulate production and post-translational modifications (PTMs) of NLRP3 inflammasome-related components (Fig. 2). Primarily, a few transcription factors, like nuclear factor kappa B (NF-κB), are triggered after PRRs recognizing stimulation by PAMPs or DAMPs [37,51,52]. Subsequently, proteins NLRP3, procaspase-1, and pro-IL-1β/18 are up-regulated in the nucleus [34,53,54]. ...
... K + efflux is a crucial mechanism behind ATP-induced NLRP3 inflammasome activation. After extracellular ATP interacts with P2X7 in LPS-primed macrophages, K + and Cl − efflux leads to the formation of NLRP3 inflammasome [37]. In macrophages, NLRP3 inflammasome is enhanced by K + efflux via the two-pore domain weak inwardly rectifying the K + channel 2 (TWIK2) channel to promote inflammation. ...
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... Again, the evidence points towards cellular energy: i) endolysosomal acidification requires ATP, ii) intracellular ATP has been shown to decrease in response to NLRP3 stimuli through K + -and Ca 2+ -mediated mitochondrial dysfunction, and iii) artificial decrease of intracellular ATP through inhibition of glycolysis has been shown to trigger NLRP3 (148). Moreover, triggering of NLRP3 through P2X 7 involves mobilisation of mitochondrial potassium (24). Recently, ATP-generation in mitochondria was found to be driven to a large extent by the secondary K + gradient (42) (generated by the mitochondrial H + /K + antiporter (58)), providing a possible mechanistic explanation to how potassium outflux could lead to a strong and immediate reduction of ATP production. ...
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The NLRP3 inflammasome is a key regulator of inflammation that responds to a broad range of stimuli. The exact mechanism of activation has not been determined, but there is a consensus on cellular potassium efflux as a major common denominator. Once NLRP3 is activated, it forms high-order complexes together with NEK7 that trigger aggregation of ASC into specks. Typically, there is only one speck per cell, consistent with the proposal that specks form – or end up at – the centrosome. ASC polymerisation in turn triggers caspase-1 activation, leading to maturation and release of IL-1β and pyroptosis, i.e., highly inflammatory cell death. Several gain-of-function mutations in the NLRP3 inflammasome have been suggested to induce spontaneous activation of NLRP3 and hence contribute to development and disease severity in numerous autoinflammatory and autoimmune diseases. Consequently, the NLRP3 inflammasome is of significant clinical interest, and recent attention has drastically improved our insight in the range of involved triggers and mechanisms of signal transduction. However, despite recent progress in knowledge, a clear and comprehensive overview of how these mechanisms interplay to shape the system level function is missing from the literature. Here, we provide such an overview as a resource to researchers working in or entering the field, as well as a computational model that allows for evaluating and explaining the function of the NLRP3 inflammasome system from the current molecular knowledge. We present a detailed reconstruction of the molecular network surrounding the NLRP3 inflammasome, which account for each specific reaction and the known regulatory constraints on each event as well as the mechanisms of drug action and impact of genetics when known. Furthermore, an executable model from this network reconstruction is generated with the aim to be used to explain NLRP3 activation from priming and activation to the maturation and release of IL-1β and IL-18. Finally, we test this detailed mechanistic model against data on the effect of different modes of inhibition of NLRP3 assembly. While the exact mechanisms of NLRP3 activation remains elusive, the literature indicates that the different stimuli converge on a single activation mechanism that is additionally controlled by distinct (positive or negative) priming and licensing events through covalent modifications of the NLRP3 molecule. Taken together, we present a compilation of the literature knowledge on the molecular mechanisms on NLRP3 activation, a detailed mechanistic model of NLRP3 activation, and explore the convergence of diverse NLRP3 activation stimuli into a single input mechanism.
... This is referred to as "macropore formation," and this enables permeation to membrane-impermeable large molecules such as ethidium and TO-PRO3 (11)(12)(13). This macropore formation and resulting K þ efflux are critical steps for the formation and activation of the nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing (NLRP)-3 inflammasome complex (14,15). Based on these backgrounds, many recent studies have focused on the mechanism of pore formation in P2X7 receptors and its downstream signaling pathways (12). ...
... In macrophages, P2X7 receptors are known to play critical roles in the formation of NLRP3 inflammasome by promoting both Ca 2 þ influx and K þ efflux (15). In the setting, macrophages secrete cleaved and matured IL-1b and promote inflammatory responses (9). ...
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The ionotropic purinergic P2X7 receptor responds to extracellular ATP and can trigger pro-inflammatory immune signaling in macrophages. Caveolin-1 (Cav-1) is known to modulate functions of macrophages and innate immunity. However, it is unknown how Cav-1 modulates P2X7 receptor activity in macrophages. We herein examined P2X7 receptor activity and macrophage functions using bone marrow-derived macrophages (BMDMs) from wild-type (WT) and Cav-1 knockout (KO) mice. ATP (1 mM) application caused biphasic increase in cytosolic [Ca ²⁺ ] and sustained decrease in cytosolic [K ⁺ ]. A specific P2X7 receptor blocker, A-740003, inhibited the maintained cytosolic [Ca ²⁺ ] increase and cytosolic [K ⁺ ] decrease. Total internal reflection fluorescent imaging and proximity ligation assays revealed a novel molecular complex formation between P2X7 receptors and Cav-1 in WT BMDMs that were stimulated with lipopolysaccharides. This molecular coupling was increased by ATP application. Specifically, the ATP-induced Ca ²⁺ influx and K ⁺ efflux through P2X7 receptors were increased in Cav-1 KO BMDMs, even though the total and surface protein levels of P2X7 receptors in WT and Cav-1 KO BMDMs were unchanged. Cell-impermeable dye (TO-PRO3) uptake analysis revealed that macro-pore formation of P2X7 receptors was enhanced in Cav-1 KO BMDMs. Cav-1 KO BMDMs increased ATP-induced IL-1b secretion, reactive oxygen species production, Gasdermin D (GSDMD) cleavage, and lactate dehydrogenase release indicating pyroptosis. A-740003 completely prevented ATP-induced pyroptosis. In combination, these data sets show that Cav-1 has a negative effect on P2X7 receptor activity in BMDMs and that Cav-1 in macrophages may contribute to finely tuned immune responses by preventing excessive IL-1b secretion and pyroptosis.